Large-scale production of human mesenchymal stem cells for clinical applications

Transcription

1 Large-scale production of human mesenchymal stem cells for clinical applications Biotechnology and Applied Biochemistry Sunghoon Jung, 1 Krishna M. Panchalingam, 1 Reynold D. Wuerth, 1 Lawrence Rosenberg, 2 and Leo A. Behie 1 1 Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada 2 McGill University, Montreal, Quebec Abstract. Human mesenchymal stem cells (hmscs) have many potential applications in tissue engineering and regenerative medicine. Currently, hmscs are generated through conventional static adherent cultures in the presence of fetal bovine serum (FBS) for clinical applications (e.g., multiple sclerosis). However, these methods are not appropriate to meet the expected future demand for quality-assured hmscs for human therapeutic use. Hence, it is imperative to develop an effective hmsc production system, which should be controllable, reproducible, and scalable. To this end, efforts have been C 2012 International Union of Biochemistry and Molecular Biology, Inc. Volume 59, Number 2, March/April 2012, Pages behie@ucalgary.ca made by several international research groups to develop (i) alternative media either by replacing FBS with human-sourced supplements (such as human serum or platelet lysate) or by identifying defined serum-free formulations consisting of key growth/attachment factors, and (ii) controlled bioreactor protocols. In this regard, we review here current hmsc production technologies and future perspectives toward efficient methods for the generation of clinically relevant numbers of hmsc therapeutics. Keywords: bioreactor, defined medium, expansion, mesenchymal stem cell, microcarrier, serum-free medium 1. Introduction Human mesenchymal stem cells (hmscs), also referred to as multipotent mesenchymal stromal cells, are present in the bone marrow (BM) and other niches of the body, and can be readily isolated and expanded through cell culture. Culture-expanded hmscs exhibit regenerative properties such as immune modulation as well as high proliferation and multilineage differentiation potential [1],[2]. These characteristics have made hmscs an attractive candidate for various therapeutic applications. Clinical studies employing hmscs have been initiated for the treatment of several diseases and injuries such as myocardial infarction, osteogenesis imperfecta, graft-versus-host disease, Crohn s disease, spinal cord injury, multiple sclerosis, and diabetes ( For therapeutic use, the extensive expansion of hmscs is critical to generate clinically relevant Abbreviations: AT, adipose tissue; BM, bone marrow; CFU-F, colony-forming unit-fibroblast; DMEM, Dulbecco s modified Eagle s medium; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; GMP, good manufacturing practice; hmsc, human mesenchymal stem cell; hmscs, human mesenchymal stem cells; MNCs, mononuclear cells; MSC, mesenchymal stem cell; MSCs, mesenchymal stem cells; OUR, oxygen utilization rate; P 0, passage zero; PDs, population doublings; PPRF-msc6, serum-free medium for hmscs; STRs, stirred tank reactors; UCB, umbilical cord blood; α-mem, alpha-minimum essential medium. Address for correspondence: Professor Leo A. Behie, PhD, PEng, FCIC, FRSC, Canada Research Chair in BioMedical Engineering, Schulich School of Engineering, University of Calgary, 2500 University Drive NW Calgary, Alberta, Canada T2N 1N4. Tel.: ; Fax: ; behie@ucalgary.ca. Received 16 November 2011; accepted 30 January 2012 DOI: /bab.1006 Published online 16 April 2012 in Wiley Online Library (wileyonlinelibrary.com) numbers of cells due to the fact that the frequency of primary hmscs is low [1]. Human mesenchymal stem cells are currently generated through conventional static adherent cultures in the presence of fetal bovine serum (FBS) or human-sourced supplements for clinical studies. However, these methods suffer from variable culture conditions (i.e., ill-defined medium components, heterogeneous culture environment, and limited growth surface area per volume), and therefore are not ideal to meet the expected future demands of quality-assured therapeutic cells for wide implementation of hmsc-related therapies. Therefore, it is desirable to develop innovative, well-characterized hmsc production processes, which should be effective for producing a large number of clinical-grade cells in a rapid, safe, and reproducible manner. These processes should be expected to offer (i) optimized growth requirements for hmscs in primary as well as passaged cultures, (ii) capacities to monitor and control culture conditions, and (iii) scalability. The growth requirements include all the extracellular nutritional, physiochemical, and mechanical factors that influence the maintenance and growth of cells in culture [3]. Below is a list of requirements for the growth of anchorage-dependent hmscs: Nutrients and regulatory factors, and their concentrations. Physiochemical environment, including dissolved oxygen and carbon dioxide concentrations, temperature, ph, osmolality, and buffer system. Culture surface. 106

2 Culture techniques (e.g., inoculation density, subculture procedures, and mechanical agitation for suspension culture). Removal or neutralization of toxic agents [i.e., negligible levels of toxins, and presence of medium components (e.g., serum) neutralizing toxic impurities]. Lack of growth-inhibitory or differentiation-inducing agents (i.e., negligible levels of factors negatively influencing cells, and suitable levels of nutrients plus regulatory factors that are otherwise toxic in excess). It is imperative to provide cells in culture with all these requirements, as precisely as possible, to ensure reproducible good growth while maintaining their regenerative and differentiation properties. Once all the requirements are identified, each of the components should be maintained within an optimal range during the entire culture period. The initial value of each extracellular variable can be changed as cells grow. For example, cells consume energy sources (e.g., glucose and glutamine) and produce metabolic by-products (e.g., lactate and ammonium), which in turn make the medium acidic. The acidic environment (i.e., low ph) is often inhibitory for cell growth. Therefore, it is critical to monitor and control the medium ph and other key variables such as oxygen tension so that they can be maintained within the values optimized for cell growth. Finally, in order to generate clinically relevant numbers of highquality cells in a timely and cost-effective manner, the production system needs to be easy to scale-up and operate. All the elements required for the large-scale production of hmscs in a controlled environment are provided from the growth medium and production system (i.e., bioreactor process). With this regard, here, we review these subjects to provide a perspective for ultimately designing an ideal hmsc production system. 2. hmsc culture Human mesenchymal stem cells are typically characterized in vitro by their (i) ability to adhere to plastic substrates; (ii) multipotency (i.e., adipogenic, chondrogenic, and osteogenic differentiation); (iii) expression of surface antigens CD73, CD90, and CD105; and (iv) lack of CD14, CD19, CD34, CD45, and HLA DR expression [4]. Although BM has been the major source of hmscs, similar cell populations have been isolated from other tissues, including adipose tissue (AT), umbilical cord tissue, umbilical cord blood (UCB), placenta, amniotic fluid, liver, lung, pancreas, and muscle [5 10]. The frequency of hmscs in BM is very low and highly variable with donor age. The colony-forming unit-fibroblast (CFU-F) assay is widely used to estimate the number of hmscs in primary BM cells and passaged cell populations in culture [11],[12]. Results from this assay indicated that cells able to generate colonies (i.e., corresponding to hmscs) comprise 1in10,000, 100,000, and250,000bmmononuclear cells (MNCs) of newborns, teens, and 30-year-olds, respectively [1]. The numbers of hmscs are also variable based on the tissue source. Kern et al. [13] reported different frequencies of hmscs in BM, AT, and UCB. In their study, with culture-initiating populations (i.e., MNCs of BM and UCB, and stromal vascular fraction of AT), it has been demonstrated that the number of CFU-Fs (i.e., corresponding to hmscs) calculated, with cells plated initially, was highest for AT (557), followed by BM (83). In contrast, the frequency of UCB was extremely low with per MNCs. Similar observations have been reported by others [14],[15]. In general, the numbers of primary hmscs, regardless of the source, are considered insufficient for research as well as clinical use. Therefore, hmscs need to be expanded through serial passages to generate clinically relevant numbers of cells (Table 1) Isolation and expansion of hmscs Universal surface markers have not been identified yet for exclusively defining hmscs, and thus these cells are typically isolated from the initial primary cell fraction (e.g., BM MNCs), based on their selective adherence to plastic substrates. Therefore, hmscs obtained are intrinsically heterogeneous, and their characteristics could be variable, depending on culture conditions. It is known that the small fraction of adherent hmscs in BM can be expanded >10 population doublings (PDs) in primary culture under classical FBS-based conditions [27]. hmscs grow at a rather constant rate during early passages (i.e., initial 3 weeks) and then the proliferation decreases gradually as the passage number increases [28]. After primary culture, hmscs Table 1 hmsc doses used for clinical trials Disease Cell mass injected References Metachromatic leukodystrophy cells/kg Koc et al. [16] Graft-versus-host disease cells/kg Le Blanc et al. [17] cells/kg Prasad et al. [18] Multiple sclerosis cells/kg Connick et al. [19] cells/patient Yamout et al. [20] Myocardial infarction cells/kg Hare et al. [21] cells/patient Chen et al. [22] Amyotrophic lateral sclerosis cells/patient Mazzini et al. [23] Osteoarthritis cells/patient Wakitani et al. [24] Spinal cord injury , , cells/patient at Park et al. [25] different locations and time points Diabetes cells/kg Jiang et al. [26] Large-scale production of hmscs 107

3 undergo >30 PDs before entering senescence [27]. Also, after the initial culture period, the multilineage differentiation potential of hmscs progressively diminishes [28] Considerations on clinical-scale hmsc production There are two distinct differences in designing bioprocesses for the production of therapeutic cells (including hmscs) when compared with conventional biopharmaceuticals (e.g., protein drugs). First, the number of therapeutic hmscs produced in one batch to treat a patient should be much smaller. Although hmscs can be expanded for >40 PDs in culture, it has been suggested that cells <20 PDs be used for clinical applications for safety and efficacy [27],[28]. For instance, from MNCs in small BM aspirates ( 2 5 ml), the expansion of hmscs (at 0.01% 0.001% of MNCs) up to 20 PDs will result in the generation of 100 1, hmscs. This was estimated by the equation, N F /N I = 2 PD, where N I and N F represent the number of hmscs plated in primary culture and harvested after 20 PDs. Therefore, the maximum number of hmscs generated in culture from a donor BM aspirate for therapeutic use should not exceed 10 9 cells for safety and efficacy. Furthermore, relatively low numbers of cells are currently being used in many clinical trials (Table 1). The scale of the bioprocess to generate these clinical doses is highly dependent upon medium formulation and bioreactor design. Under an optimized culture environment, a representative single-clinical dose ( hmscs per patient) can be produced even by using a 1 L bioreactor (discussed later). Second, unlike cell culture-derived protein drugs, hmscs represent the therapeutic products themselves, not the production tools. Therefore, it is crucial to produce healthy hmscs that retain their desired therapeutic properties. To achieve this, it is critical to identify a well-defined culture environment (i.e., all the nutritional, physiochemical, and mechanical requirements) supporting the growth of therapeutic hmscs and to maintain this optimized condition using an online control system during the entire culture period. That is, in designing a bioprocess for the production of therapeutic cells (as opposed to protein drugs), the stringent consistency in generating cells retaining regenerative properties in a safe and efficient manner is imperative. Variable cell qualities may cause treatment failures. In contrast, cellular variations in protein production can merely lead to variable product yields. Considered together, studies on designing hmsc production systems should be directed toward a well-defined, onlinecontrolled bioprocess to provide cells with an optimized growth environment on a continuous basis in a relatively small scale, that is, 1 5 L depending on medium formulation and bioreactor design. 3. hmsc growth media 3.1. Classical FBS-based media Conventional media used for isolating and expanding hmscs consist of a defined basal medium [e.g., Dulbecco s modified Eagle s medium (DMEM) or alpha-minimum essential medium (α-mem)] supplemented with FBS at 10% 20% (v/v). FBS provides a high content of growth-stimulatory factors as well as nutritional and physiochemical compounds required for cell maintenance and growth. In addition, FBS plays a role in providing essential attachment factors to facilitate cell adherence to a culture substrate, which is a prerequisite for the growth of adherent hmscs. Although FBS-based media remains the standard in generating hmscs for basic research and clinical studies, concerns have been raised due to the potential problems associated with FBS, including safety issues [29],[30]. Despite strict selection and testing for safety and growth-promoting capacity, FBS is inherently unsafe and risky because it may still contain harmful contaminants such as prion, viral, or zoonotic agents. Moreover, hmscs grown in FBS-containing media become associated with FBS proteins [31], which may cause immune reactions in patients, particularly when multiple doses are required. Furthermore, the poorly defined nature of FBS, together with its high degree of batch-to-batch variation, can cause inconsistencies in the generation of quality-assured cells. Thus, the use of FBS represents a major obstacle for the wide implementation of hmsc-related therapies Humanized media To mitigate the safety and regulatory concerns raised by animal serum, human blood-derived materials such as human serum and platelet derivatives are currently under investigation for their clinical utility as an alternative medium supplement (reviewed in [32]). Human autologous serum has been reported to support hmsc expansion [33 35]. It would be problematic, however, to acquire amounts sufficient to generate clinically relevant numbers of hmscs. The performance of human allogeneic serum is rather controversial because contradictory results have been reported [34],[36 39]. Also, there has been much evidence that human platelet-derived supplements such as platelet lysate or platelet-rich plasma have considerable growth-promoting properties for hmscs, while maintaining their differentiation potential and immunomodulatory properties [40 48]. Although considered relatively safer than FBS for human therapeutic applications, the use of human-sourced supplements is still a matter of substantial debate, prompting some concerns [49],[50]. There is a risk that allogenic human growth supplements may be contaminated with human pathogens that might not be detected by routine screening of blood donors. Moreover, these crude blood derivatives are poorly defined and suffer from batch-to-batch variation, and thus their ability to maintain hmsc growth and therapeutic potentials could be widely variable. In particular, the variability can be a significant hindrance for implementing the clinical-scale production of hmscs simply because it could make it difficult to obtain cells retaining desired qualities in a consistent and predictable manner, which is crucial for minimizing treatment failures Defined serum-free media Most of the defined serum-free media developed for animal or human MSC growth have demonstrated only limited performance [51 53]. In particular, designing a medium formulation to maintain the attachment and growth of primary hmscs has long been a challenge. Recently, our group reported the first 108 Biotechnology and Applied Biochemistry

4 defined serum-free medium formulation (PPRF-msc6) that supports the attachment and expansion of hmscs in primary culture of BM MNCs and subsequent passages, while maintaining their immunophenotype and multipotentiality [54]. Although PPRFmsc6 still contains native proteins purified from human serum (albumin and transferrin) and FBS (fetuin), which are often associated with impurities, this medium represents the most welldefined serum-free formulation to support both the isolation and expansion of hmscs in the literature. It is well known that exogenous matrix proteins (e.g., fibronectin) may be needed to precoat a culture surface to facilitate the attachment of certain anchorage-dependent cell types onto the substrate when serum-free media are used. We observed that PPRF-msc6 supports the attachment and growth of hmscs on tissue culturetreated surface, without the use of a coating material. However, the growth rates/yields were found to vary when culture vessels from different manufacturers were used [our unpublished data]. This variability was greatly reduced by coating the substrates with attachment proteins such as gelatin or fibronectin, and thus it appears to be desirable to precoat the culture surface [or add such protein(s) to the medium] to obtain optimal and reproducible hmsc growth in PPRF-msc6. In addition to the better defined nature, PPRF-msc6 has also demonstrated significantly enhanced performance on hmsc production compared with classical serum-supplemented media (e.g., DMEM supplemented with prescreened FBS; 10% FBS DMEM). Specifically, the use of PPRF-msc6 led to the formation of well-developed colonies in the primary culture of BM MNCs at an early time and higher frequencies compared with 10% FBS DMEM (Fig. 1). This ability was very consistent with multiple donor BM cells, resulting in the recovery of significantly higher numbers of passage zero (P 0 ) cells [55]. Also, PPRF-msc6 sustained this en- Fig. 1. Colony-forming unit-fibroblast assay of primary human bone marrow mononuclear cells (BM MNCs) using a classical FBS-based medium (A) and a defined serum-free PPRF-msc6 medium (B). Photos show colonies developed for 12 days at different seeding densities of BM MNCs as indicated that is, 90,000, 180,000, and 360,000 BM MNCs per well (9.6 cm 2 in area). Adapted with permission from S. Jung s PhD Thesis, ref. [58]. hanced growth through serial passages, demonstrating significantly lower cell doubling times and greater PDs. Furthermore, the use of this serum-free medium generated a more homogeneous hmsc population, which was smaller in size than those expanded in 10% FBS DMEM. The size of cells to be administered to patients could be an important issue because it has been shown in animal model studies that most of hmscs grown in FBS-supplemented media were trapped in the lung [56]. Small hmscs may offer a significant benefit in transplantation therapies because the small cells may travel through the lung and home to the site of injury or disease at high efficiencies [57]. From a production viewpoint, the production of smaller cells requires less surface area and thus reduced number or volume of culture vessels, reducing risk of contamination and saving cost and labor. PPRF-msc6 also supported the derivation and serial expansion of hmscs from AT and pancreas up to over 90 and 70 PDs, respectively, within 3 months [our unpublished data]. Several commercial serum-free media have recently been developed for the expansion of hmscs (reviewed in [59]). Invitrogen (Burlington, Ontario, Canada) introduced the first commercial serum-free medium, STEMPRO R MSC SFM, demonstrating that this medium supported hmsc growth more efficiently compared with a control FBS medium. Later, the company manufactured a revised xeno-free version of this medium, and other companies also introduced xeno-free, serum-free media for hmsc culture. All these products were reported to support hmsc expansion, and thus may be useful for therapeutic applications once their efficacy and safety are proven. However, the formulations of these commercial media are not disclosed, which may restrict their wide utility in hmsc research and clinical studies. Recently, STEMPRO R MSC SFM has been cleared by the US Food and Drug Administration as a medical device for clinical trials in the United States ( We evaluated and compared the performance of this serum-free commercial medium to PPRF-msc6 and 10% FBS DMEM for hmsc growth. When primary BM MNCs were plated into human fibronectincoated tissue culture flasks containing each of these three media, the rate and pattern of colony formation were quite different from each other. Mature (i.e., large and dense) colonies were generated in PPRF-msc6 at day 11 after inoculation, whereas colonies in both 10% FBS DMEM and STEMPRO R MSC SFM were still immature (Figs. 2A 2C). The immature colonies grown in 10% FBS DMEM were further allowed to grow until they became large and dense (on day 14). The cells in STEMPRO R MSC SFM did not generate well-developed colonies, although they were allowed to grow for a longer period of time (up to day 17), but started to form clusters. The primary culture using PPRF-msc6 was harvested earlier (on day 11) as a large number of well-developed colonies were formed. Nonetheless, the cell yield was still significantly higher than those obtained in both 10% FBS DMEM and STEMPRO R MSC SFM, which were harvested on days 14 and 17, respectively (Fig. 2D). The higher growth rate in PPRF-msc6 was sustained through serial passaging until the cells entered senescence (Fig. 2E). Taken together, based on the beneficial features (i.e., better defined nature, high-growth-promoting performance, and generation of small size of cells), PPRF-msc6 represents a Large-scale production of hmscs 109

5 Fig. 2. Primary and passaged cultures of hmscs in three different media. Photomicrographs (at 5 ) show representative colonies in PPRF-msc6 (A), 10% FBS DMEM (B), or STEMPRO R MSC SFM (C) at the time points indicated. (D) Cells in each condition were harvested to determine cell yield when well-developed colonies appeared at different time points indicated. (E) The recovered cells from the primary culture were serially passaged in respective medium. Adapted with permission from S. Jung s PhD Thesis, ref. [58]. significant step forward for producing hmscs in an efficient and consistent manner. The protocol for PPRF-msc6 preparation has been described in detail [54], so that the disclosed formulation or its modifications can be further exploited. 4. Bioreactors for the expansion of hmscs In addition to the requirement of a defined serum-free medium, the production of therapeutic cells in accordance with good manufacturing practice (GMP) requires a scalable and controllable bioprocess that can be operated in a closed system. The most widely used cell culture bioreactor in a laboratory scale, in particular for anchorage-dependent cells, is the T-flask with various surface areas that is, 25, 75, 150, and 225 cm 2 T-flasks. It is easy to use, cost-effective, and disposable. T-flasks with filter-coupled caps allow for the exchange of gases (i.e., oxygen and carbon dioxide). However, the generation of a large number of cells requires manipulating numerous vessels, which is time consuming and labor intensive and tends to result in vessel-tovessel variability in cell growth and quality. Handling multiple vessels also increases the possibility of contact with external contaminants. Moreover, this culture system is not appropriate for controlling process parameters due to the heterogeneous nature of the culture environment. Despite its usefulness in basic research, therefore, the utility of T-flasks is not efficient 110 Biotechnology and Applied Biochemistry

6 for the generation of clinically relevant quantities of uniform hmscs. For the scalable expansion of hmscs, various types of bioreactors have been used, including multilayered cell factories, roller bottles, stirred bioreactors (with microcarriers), hollow-fiber bioreactors, and fixed-bed bioreactors (reviewed in [60 63]). Each bioreactor design has its own specific features, and thus it is important to evaluate and compare different bioreactors in order to select the best type for the large-scale production of quality-assured cells. It should be pointed out, however, that it is not easy to compare different types of bioreactors because cell growth requirements (nutritional, physiochemical, and mechanical) may be different according to the bioreactor design. Therefore, the performance of each bioreactor system should be optimized prior to being compared with others. For example, early attempts showed that stirred suspension cultures using microcarriers exhibited significantly lower growth rates of human and animal MSCs when compared with control static cultures [64 66]. However, recent efforts enhanced the performance of microcarrier culture by investigating the effect of medium components and other culture variables [67],[68]. These improvements suggest that the microcarrier technology platform could be a realistic option for the next generation of scaled-up hmsc production systems. Beyond the performance of bioreactors for supporting good growth and maintenance of therapeutic properties of cells, specific features of different bioreactor types should also be considered from a practical standpoint, comparing the advantages and disadvantages. Bioreactor-specific characteristics that need to be considered are as follows: Simplicity of operation. Attainable cell density. Disposability. Ability to incorporate online monitoring and control. Automation. Ease of harvest. Effectiveness in terms of cost and time. The selected bioreactor system must be easy to operate and achieve high cell densities. Disposable bioreactors are preferable, if cost-effective, due to the elimination of cleaning and sterilization steps. Online monitoring and tight control of the culture system are critical to maintain environmental variables within ranges established for optimal cell growth. This can be readily achieved with bioreactors in which cells grow in uniform conditions. Suspension bioreactors facilitate the process automation, which could considerably save labor and time and thus production cost. As the anchorage-dependent hmscs should be separated from the substrates (i.e., flat surface, hollow fibers, microcarriers, and other nonporous and porous immobilizing materials), a bioprocess enabling easy harvesting is desirable. In this section, we review briefly simple bioreactors that are currently used for the production of hmscs, and discuss the features of the stirred microcarrier culture system and the important considerations to achieve its high and consistent performance. Other bioreactor designs, including the hollow-fiber bioreactor [69] and fixed-bed bioreactor [70], are not discussed in this report. The widespread implementation of hmsc-related therapies must require the development of efficient culture systems. The ideal process should be a GMP-compliant, closed system, which is characterized to be robust, scalable, controllable, and easy to operate, supporting the large production of quality-assured hmscs in a timely and cost-effective manner Multilayered cell factories The cell factory is an extended version of T-flask configuration, representing the simplest system for scaling up monolayer culture. This multilayered culture system is designed to offer a large surface for cell growth by building up many stacks in a unit vessel. Typically, each unit consists of 1 10 stacks that are connected together; if necessary, extra layers can be added. Therefore, the number of culture vessels can be decreased by using multilayered cell factories, which may reduce vessel-to-vessel variability. Probably the most attractive aspect of using the cell factories is the fact that they have a similar nature of geometry and substrate with T-flasks, and thus the technical translation from the T-flask culture to the multilayered culture is straightforward. For this reason, this method has been used for generating hmscs by many investigators [19],[47],[71 73]. Bartmann et al. [71] reported that the growth of hmscs was not changed in cell factories (four layered) compared with T-225 flasks. To obtain a clinical dose of hmscs (> cells), four to 10 four-layered cell factories were used [71],[73]. Although easy to implement, this approach is costly in time and money and difficult to monitor and control the culture. Moreover, this process suffers from the potential vessel-to-vessel variability and the difficulty of scale-up, and thus is not effective if higher doses of cells are demanded. Furthermore, the recovery of all the cells from the vessel after trypsinization could be problematic Roller bottles Roller bottles are cylindrical vessels made of tissue culture plastic or glass, which are placed on their side onto a roller rack that rotates them gently. During the rotation, the medium carrying inoculated cells moves continuously over the inner surface to allow cell attachment, and the cells grow to form monolayer over nearly all the surface. Therefore, a greater surface area for growth per vessel is offered compared with T-flasks (Table 2), while this process provides fundamentally the same culture techniques. In addition, in the rotated bottles, the cell layer is exposed to medium, which covers only 15% 20% of the surface, and air alternatively. This mechanism allows for smaller amounts of medium to be used and results in an increased rate of mass transfer through the thin liquid film over the cells during their transient exposure to the gaseous phase in the bottle. Moreover, in contrast to static cultures in which concentration gradients of nutrients and physiochemical variables in stagnant medium (e.g., dissolved oxygen and ph) occur, the gentle movement reduces such gradients so that cells are exposed to a relatively uniform microenvironment. The disadvantages of this process is that it requires a roller rack to rotate the cylindrical vessels and the procedure is labor intensive as each bottle should be handled individually for cell Large-scale production of hmscs 111

7 Table 2 Surface area of anchorage-dependent culture vessels Culture type Surface area per unit Working volume Equivalent surface areas Surface area (cm 2 ) per liter Cytodex 1 4,400 cm 2 /g Same as the vessel size 1 L stirred vessel (1 5 g/l) 4,400 22,000 Cytodex 3 2,700 cm 2 /g Same as the vessel size 1 L stirred vessel ( g/l) 4,400 22,000 Roller bottles 850 or 1700 cm 2 /bottle ml/850 cm cm 2 bottles 3,333 5, ml/1700 cm cm 2 bottles Cell factories 632 cm 2 /layer 200 ml/layer 7 35 layers 3,160 T-flasks 225 cm 2 /flask 71 ml/flask flasks 3,160 inoculation, harvest, and medium feeding. Also, online monitoring and control of environmental variables is difficult. However, despite these drawbacks, roller bottles are most commonly used for the initial industrial-scale production of biopharmaceuticals as they are easy to implement and automatic/robotic operations can be designed to handle a large number of vessels [74],[75]. Roller bottles have been used for MSC-based tissue engineering applications [76],[77], and recently for the expansion of hmscs and hematopoietic cells [78],[79]. Cohen and McNiece [79] demonstrated enhanced expansion of hmscs in roller bottles with no significant changes in morphology, but reduced expression of interleukin-1 and interleukin-6, compared with those grown in T-flasks. Therefore, investigations need to be carried out to further characterize hmscs grown in roller bottles and to examine the effect of intrinsic culture variables on cells (e.g., rotating speed). Although roller bottles have not yet been widely used for hmsc expansion, the good features of this system as described earlier and the extensive industrial experience may render this system effective for the production of hmscs in an uncomplicated manner Microcarrier-based stirred bioreactors Stirred bioreactors, such as spinner flasks and stirred-tank reactors (STRs), are cylindrical vessels with an impeller. These systems provide relatively uniform conditions throughout the vessel due to mixing induced by an impeller. The impeller is rotated by a magnetic stirrer or by a top-driven motor. Moreover, these stirred bioreactors can be easily operated and scaled-up, and thus the generation of a desired number of cells can readily be achieved using a single vessel at different scales (Table 2). This avoids vessel-to-vessel variability and saves on time and labor. Furthermore, these processes can be readily equipped with computer-controlled online monitoring instruments, which taken together with the well-mixed, homogeneous environment enables tight control of process variables such as ph, temperature, and dissolved oxygen and carbon dioxide concentrations. The ability to operate this process in multiple modes (batch, fedbatch, or perfusion) also allows for the implementation of welldesigned feeding strategies that can ensure an adequate supply of required medium components and reduce levels of waste metabolic products (e.g., lactate and ammonium) that may have an adverse effect on cell proliferation or cause undesired differentiation. On the basis of these advantages, stirred bioreactors have been widely used to culture anchorage-dependent cells (with microcarriers) as well as suspension cells. Microcarriers are microscopic beads (with a diameter of μm) that can be placed in medium to provide a surface upon which anchoragedependent cells can attach and subsequently grow under pseudosuspension conditions [74],[75],[80]. Depending on the loading density of microcarriers in culture, this method can provide a significantly high ratio of the growth surface to medium volume. Therefore, microcarrier cultures in stirred bioreactors could offer a superior means of producing large quantities of adherent cells. Microcarriers have also been used as substrates for hmsc culture or tissue engineering applications (reviewed in [62],[63]). Early efforts examining the effect of microcarriers on MSCs derived from animals and humans were primarily made for tissue engineering. In these studies, cells seeded onto plastic, hydroxyapatite, or calcium titanium phosphate microcarriers were cultured in tissue culture plates [81],[82], rotating bioreactors [83],[84], or spinner flasks [85] for osteogenic differentiation. On the basis of a cell production viewpoint, the first attempt to use microcarriers for the expansion of MSCs was made by Frauenschuh et al. [64] using porcine BM cells. This study was followed by efforts made by other investigators, using MSCs derived from various animal and human sources such as BM, placenta, and ear [65 68],[86 89]. Even though these studies demonstrated the possibility of using microcarrier-based suspension culture for hmsc expansion, the performance of this approach is relatively low and highly variable, compared with conventional static cultures. Therefore, further efforts need to be made to bring this technology to a reality for the robust and reproducible production of hmscs. Designing an optimal culture system for a specific cell type depends on understanding the characteristics of the cell and the growth requirements. In addition to the nutritional and physicochemical environment in static cultures, fluid mechanics also affect cells in stirred bioreactors. It is very important to keep in mind that with the use of microcarriers, the dynamic culture system is quite complex, having a large number of key variables. For this reason, although the microcarrier technology has a number of advantages over static cultures for large-scale biological production, most cell culture processes still employ simple systems such as roller bottles [90]. Moreover, hmscs are highly sensitive to shear stress induced by mechanical agitation. Therefore, to achieve a successful, consistent large-scale production of these fragile cells, each of the parameters critical for the microcarrier-based stirred cultures should be recognized, optimized, and standardized. Below is a list of the variables that should be considered: 112 Biotechnology and Applied Biochemistry

8 Selection of microcarriers (chemical, physical, and geometrical properties). Microcarrier loading density. Cell seeding density. Medium feeding (particularly for high-density culture). Medium composition (attachment and growth factors, shearprotecting agents). Substrate-coating material (particularly for serum-free culture). Stirring protocol (i.e., impeller tip speed, continuous vs. intermittent agitation for initial cell attachment, variation of agitation rate according to cell growth). Bioreactor shape, configuration, and treatment. Treatment of the surface of vessel, impeller, and other inserts to prevent cell attachment (e.g., siliconization). Geometry, size, and location of impeller and other instruments submerged in culture. Aeration protocol (i.e., headspace aeration vs. sparging). Additional parameters for cell attachment phase. Initial culture volume. Initial ph. Initial medium composition (e.g., serum content). Preparation of microcarriers and cell inocula. Bead-to-bead transfer. Cell harvesting protocol Selection of microcarriers The selection of microcarriers for MSC culture has been examined by several investigators. Frauenschuh et al. [64] demonstrated that Cytodex type 1 microcarriers led to better adherence of porcine MSCs compared with Cytodex 2 and 3 microcarriers. Schop et al. [66] also reported that the microporous Cytodex 1 microcarriers showed higher cell attachment efficiency for hmscs from BM in comparison with Cytodex 3 and other solid microcarrier types made of different materials. In contrast, we found that Cytodex 3 was superior to Cytodex 1 in terms of attachment as well as growth of BM hmscs in our experiments [91]. Whereas Schop et al. [66] performed the attachment efficiency test using 24-well plates, our group conducted the microcarrier cultures in spinner flasks. Moreover, different medium formulations were used in both studies (i.e., 15% FBS α-mem with additional supplements [66] and 10% FBS DMEM [91]). Therefore, it is difficult to compare the data reported by these two groups, although both studies used the same cell type. It has also been reported that macroporous microcarriers, such as Cultispher-S, could be used for the cultivation of MSCs [67],[68],[80],[86],[87]. In contrast to microporous microcarriers, which were designed to offer their external surface for cell growth while allowing nutritional transportation through microscopic pores, macroporous microcarriers were originally developed to support higher cell density cultures by providing internal space and thus a larger surface for cell growth. That is, the pores of each macroporous microcarrier bead are large enough for cells to migrate into and grow. This may provide a benefit for MSC culture as the fragile cells growing inside beads may be protected against detrimental shear stress. Recently, animal component-free, nonporous plastic microcarriers have also been successfully used for hmsc culture in spinner flasks [67] Microcarrier and cell seeding density Microcarrier and cell seeding densities and cell-to-bead ratio are well-known factors influencing (i) culture efficiencies based on the initial event of cell attachment to microcarriers and (ii) the level of culture compactness. The attachment of cells to microcarriers at inoculation occurs according to a probability distribution, and thus it is important to seed cells and microcarriers at an optimal ratio to achieve a good cell-to-bead distribution in which each bead is occupied by at least one viable cell. For example, given that cell attachment follows a Poisson process [64], the theoretical probabilities of unoccupied microcarriers at a cell-to-bead ratio of 1, 2, 3, and 4 are 0.365, 0.135, 0.05, and 0.018, respectively. In addition, these probabilities are likely to be decreased under suboptimal inoculation conditions (e.g., inhibitory medium components, suboptimal microcarrier type, cellular damage, or adverse ph). Therefore, cells and microcarriers are generally seeded with a minimum inoculation level of 3 or 4 cells per bead [92]. Microcarrier concentrations used for most mammalian cell cultures are within a range of 1 3 g microcarriers (dry weight) per liter. A typical final cell concentration maintained in this condition is known to be about cells/ml for most mammalian cell lines [74]. In contrast, the final concentrations of hmscs obtained from fed-batch spinner cultures with a common microcarrier loading density (i.e., 2 g/l) were reported to be in a range of cells/ml, depending on medium formulations and cell source [67],[68]. The difference in densities of hmscs and other cell lines obtained at the end of culture may be due to, but not limited to, suboptimal culture conditions for hmscs and/or their larger cell size. To increase the hmsc yield per volume, higher microcarrier loading densities could be used; however, this requires high numbers of cell inocula to meet optimal cell-to-bead ratios, and the resulting dense cultures may render proper culture stirring difficult. Moreover, the high-density cultures may need to involve air sparging and frequent medium feeding to supply cells with adequate oxygen and other nutrients, which will make the process operation more complicated Medium feeding High-density cultures using microcarriers, in particular with relatively high loading concentrations, are likely to cause premature exhaustion of key nutrients and/or accumulation of toxic metabolic products such as ammonium and lactate, and thus a well-established feeding strategy may be necessary. In this regard, it is important to examine the metabolic profiles during the culture and perform medium replenishment accordingly in order to avoid growth inhibition due to nutrients and metabolites [66],[68],[87]. Also, it was reported that hmsc metabolic profiling in microcarrier-based stirred cultures was different from that observed in static conditions, presumably due to the metabolic adaptation to the dynamic environment [66],[67]. Therefore, to establish a well-designed optimal feeding protocol, it is desirable to obtain metabolic information directly from the Large-scale production of hmscs 113

9 findings suggest that, beyond the safety issues, the development of a good medium is a key component in designing a high-performance bioprocess for scalable hmsc production. Fig. 3. Expansion of hmscs in PPRF-msc6 versus 10% FBS DMEM in stirred suspension culture using Cytodex type 3 microcarriers. While the FBS-supplemented medium resulted in a considerably prolonged lag phase and a slow cell growth, PPRF-msc6 led to a diminished lag phase and a higher cell expansion (>18-fold expansion without medium change). microcarrier culture, not by indirect interpretation of data from static culture Medium formulation The cultivation of animal and human MSCs in microcarrier cultures using conventional serum-based media exhibits a prolonged lag phase and a low growth rate [64],[66],[68]. Minimizing the duration of the lag phase and maximizing the rate and length of the exponential growth phase are requirements in designing a good bioprocess. We recently demonstrated that by identifying key growth and attachment factors for hmscs and designing a serum-free medium formulation (PPRF-msc6), we were able to significantly reduce the lag phase and increase the growth rate of hmscs in static culture [54],[55]. We further tested PPRF-msc6 medium for the cultivation of hmscs in microcarrierbased spinner flasks in comparison with 10% FBS DMEM, and found that the superior performance of PPRF-msc6 observed in the static cultures was also evident in this stirred culture system (Fig. 3). Specifically, when hmscs were inoculated at cells/ml into spinner flasks containing 2 g/l of Cytodex 3 microcarriers (precoated with FBS discussed later), a maximum cell density of ± cells/ml was obtained using PPRF-msc6 medium on day 6.5 (total ± 0.94 cell-fold expansion, and an average of 2.81-fold expansion per day), showing a considerably reduced lag phase and increased growth rate compared with the serum-based culture. In contrast, the use of 10% FBS DMEM resulted in a maximum cell density of ± cells/ml on day 11 (i.e., total 8.66 ± 1.01 cell-fold expansion, and an average of 0.79-fold expansion per day). Similarly, Eibes et al. [68] reported that a commercial medium containing a lower amount of serum significantly enhanced hmsc expansion compared with 10% FBS DMEM. These Substrate-coating material Eibes et al. [68] reported that, when nonplastic microcarriers were precoated with a commercial substrate-coating material, only 22% of hmscs inoculated adhered to microcarrier beads in a serum-free medium, which resulted in a considerably delayed cell expansion. In contrast, the use of same or similar serum-free media together with the same coating material maintained a high initial attachment of hmscs from BM and AT in static cultures [93],[94]. Similarly, we observed that, although PPRF-msc6 medium supported good initial attachment of hmscs onto a substrate coated with a defined coating protein such as fibronectin, gelatin, and collagen in static cultures, the serum-free medium led to poor cell attachment onto collagen Cytodex 3 microcarriers in a stirred environment. For this reason, we and others precoated microcarriers with FBS prior to serum-free or serum-reduced hmsc cultures, and this resulted in efficient cell adherence [67],[68]. From a therapeutic viewpoint, the use of FBS to coat microcarriers is not desirable. Therefore, in addition to the selection of an appropriate microcarrier type, key coating materials and/or medium components to facilitate cell attachment to the beads should be identified in order to improve the microcarrier culture efficiency, particularly for serum-free cultures Bioreactor configuration and mixing protocol The hydrodynamics in bioreactors is influenced by factors related to bioreactor configuration and mixing, including (i) vessel shape, (ii) impeller (geometry, size, location, and speed), and (iii) probes and other instruments (e.g., baffles) submerged in culture (shape and location) [61]. In stirred microcarrier cultures, cellular damage is caused mainly by shear forces induced by the interaction of microcarrier beads with small turbulent eddies and by bead-to-bead collisions occurring in the hydrodynamic field [95]. Therefore, designing an optimal bioreactor configuration and mixing protocol is critical to minimize cell death and growth inhibition. The bottom of bioreactor vessels should be curved for better mixing and have a gentle dimple in the middle to minimize a dead zone that could be formed below a suspended impeller. Furthermore, the electropolished internal surface of the vessels may need to be treated with silicon to prevent attachment of microcarriers and cells to the inner wall. Selection of an impeller type that is best suited for the process is crucial. In general, pitched-blade impellers with flat blades at 45 are known to be suitable for the cultivation of fragile mammalian cells on microcarriers in stirred bioreactors because this type of impeller causes less shear and offers good mixing by producing an axial and radial flow simultaneously [96]. However, we observed that a commercial pitched-blade impeller poorly supported hmsc expansion. In contrast, our custom-designed four-blade impeller, largely generating a radial flow, greatly increased expansion [our unpublished data]. This observation indicates that the selection of impeller type 114 Biotechnology and Applied Biochemistry

10 should be cell-type specific, and thus further studies to identify the best impeller configuration need to be conducted. The size of impeller (in the ratio to vessel size) is also important. Larger impellers could support a uniform condition at lower speeds, inducing lower shear forces. The presence of probes and other submerged parts that perturb the liquid flow also influences shear forces. Therefore, these instruments as well as the impeller should be located at appropriate positions, and this configuration should be kept constant for each bioreactor run in order to maintain a consistent performance. Stirring protocols should be optimized for maximizing initial cell-to-microcarrier interactions as well as growth. It is well recognized that intermittent stirring regimes along with reduced culture volume may facilitate cell attachment during the initial phase, and thus various protocols have been employed to enhance the initial attachment efficiency of MSCs and similar populations [64],[67],[97]. During the expansion phase, the stirring process should be gentle to minimize detrimental shear forces [95], while being sufficient to maintain well-mixed uniform conditions. Specific gravity of most microcarrier beads used in stirred bioreactors varies from 1.02 to 1.04 g/cm 3 and influences the stirring speed, as higher density beads require a higher speed [75]. Also important, as the cultures progress, the degree of cell growth and bead agglomeration will greatly increase the sedimentation velocity of the cell microcarrier complexes. Therefore, the stirring speed may need to be tailored at different time points to maintain a homogeneous culture environment Aeration protocols Adequate oxygenation to support cell metabolism is one of the major issues associated with the bioprocess scale-up. If direct air sparging is used for oxygen supply, the rising gas bubbles may produce strong hydrodynamic forces, causing cell death and bead agglomeration at the air medium interface [95]. Oxygen supply by diffusion from the cylindrical bioreactor headspace through the culture surface may be sufficient for the clinical-scale production of hmscs (< L). In culture, if cells utilize oxygen faster than it can be supplied, the dissolved oxygen concentration in the medium will eventually decrease to a level that may not support cell growth. To prevent this, the oxygen transfer rate across the medium surface must be increased above the oxygen utilization rate (OUR) of the cells. It is known that the OUR of mammalian cells varies from 0.06 to 0.6 mm/h in cultures containing cells/ml, and oxygen supplied by headspace aeration will not become limiting up to 1 L of culture in a cylindrical-shaped suspension bioreactor at this cell density. For the surface aeration, the ratio of culture surface area to height should be at least 1:1 [98]. Therefore, to be able to use surface aeration without oxygen depletion, the maximum culture volume should be estimated accordingly Initial condition for facilitating cell attachment Optimizing the inoculation condition for maximizing cell attachment to microcarrier beads with a good distribution is critical for a successful microcarrier culture. Cell attachment to the microcarrier surface is a physical process involving van der Waals interactions, and this event is influenced by many variables, including microcarrier surface properties, medium ph, and medium components, as well as stirring protocol [92]. Forestell et al. [92] enhanced attachment efficiencies of human fetal lung fibroblasts on Cytodex 1 microcarriers by lowering ph (7.0) and serum concentration (4%) during an initial period of 3 H as opposed to the optimal growth condition (ph 7.7 and 10% serum). Moreover, the use of a low serum medium (i.e., 2% FBS) significantly reduced the initial lag phase in hmsc culture using macroporous microcarriers (Cultispher-S) compared with classical 10% FBS DMEM [68]. Our serum-free PPRF-msc6 medium also led to considerably reduced lag time in Cytodex 3-based hmsc culture as shown in Fig. 3. The initial lag phase was almost negligible in static cultures using PPRF-msc6 [55]. Therefore, optimizing the inoculation condition in the microcarrier-based stirred cultures for hmscs with PPRF-msc6 may further diminish the lag phase Bead-to-bead transfer Increasing cell yields in microcarrier culture could be achieved by bead-to-bead transfer, avoiding the need to dissociate cells from the beads. This can be performed by adding fresh microcarriers into an existing culture containing old microcarriers loaded with cells so that some of the cells migrate over and begin to grow on the empty beads. The bead-to-bead transfer has been employed for the cultivation of MSCs [64 66],[86] as well as other cell types [99],[100]. Although this technique could be exploited as an efficient operation for the scaled-up production of hmscs, it may lead to a more complex process because the bead-to-bead transfer event needs to occur efficiently. Several protocols have been suggested to facilitate the transfer for example, intermittent stirring, partial dissociation of cells on the original beads, use of low calcium medium, and ph change [74],[99],[100] Cell harvest protocol Cell harvesting techniques are imperative in preparing healthy hmscs to be used for their therapeutic applications or for passaging, if necessary, to efficiently initiate a larger scale production system. Typically, cell bead complexes are treated with proteolytic enzymes, and the detached cells are then separated from microcarriers by filtering through a nylon mesh of appropriate size. The most commonly used method to separate MSCs from microcarrier beads includes the use of trypsin alone or in combination with chelating agents [e.g., ethylenediaminetetraacetic acid (EDTA)]. Unlike monolayer cells in static cultures, dissociating cells from microcarriers maintained in a dynamic environment is somewhat tricky and cell detachment efficiencies often vary. In several studies for MSC culture in stirred conditions, cells on microcarriers were treated with trypsin at a high concentration or for a relatively long period of time [64],[66],[67]. However, this may cause significant cell damage and phenotypic change. For instance, when cells grown in monolayer were treated with 0.25% trypsin EDTA solution for 5, 30, and 90 Min at room temperature, CD105 expression was decreased with time [101]. Other proteolytic enzymes, such as collagenase and dispase, were also used to harvest hmscs by digesting Large-scale production of hmscs 115

11 macroporous microcarriers [86],[102]. It is crucial to identify an optimal enzymatic protocol (i.e., enzyme, chelating agent, treatment temperature, and time), depending on the microcarrier type used, to maximize the quantity and quality of cells at harvest. In recent studies by Sart et al. [86],[87], dispase was initially used to dissociate rat MSCs from Cultispher-S microcarriers but later replaced by trypsin. A recombinant trypsin was also used to detach hmscs from nonporous plastic microcarriers treated with a commercial coating material [67]. As a nonenzymatic microcarrier process, Yang et al. [88] fabricated thermosensitive microcarriers to grow hmscs and then separate them from the microcarriers by decreasing culture temperature below 32 C. In their study, the surface of Cytodex 3 microcarriers was incorporated with a thermosensitive polymer, poly-n-isopropylacrylamide, and >82% of cells were detached by temperature change without the use of a proteolytic enzyme. Importantly, the cell detachment by temperature change reduced apoptosis and cell death. This technology may suggest an effective microcarrier-based cell culture process to produce healthy hmscs. 5. Generation of clinical doses of hmscs using current technologies The optimal dose of hmscs still needs to be assessed for each specific therapeutic application. Once a target dose and a treatment time schedule are determined, it is crucial to create a well-designed plan to (i) obtain a starting cell/tissue source and (ii) produce the desired number of hmscs, according to a predictable timeframe, for a patient to be administered with fresh cells on time. To this end, once a bioprocess protocol is selected including medium, bioreactor type, and operating parameters the size (i.e., the volume and number of vessels) of the bioprocess and the processing time schedule need to be estimated as precisely as possible, based on the data available for hmsc production using the same protocol. Table 3 demonstrates the estimated scales and times of culture for producing a clinical dose of hmscs (e.g., >200 million cells per patient) starting with small BM aspirates using three different methods 10% FBS DMEM + T-flasks, PPRFmsc6 + T-flasks, and PPRF-msc6 + Cytodex 3 microcarriers in computer-controlled stirred bioreactors. All the estimations were derived from our experimental data. A brief description of the bioprocess is as follows: Starting material: Small BM aspirates (2 5 ml, BM MNCs). Bioreactors: 225 cm 2 T-flask (NUNC, Roskilde, Denmark) coated with human fibronectin, 250 ml glass spinner flask (Corning, Corning, NY, USA) with a two-blade magnet impeller (without computer control capabilities), and 500 ml glass STRs (DASGIP Bioreactor, Julich, North Rhine Westphalia, Germany) with a four-blade magnet impeller and associated probes (with computer control capabilities). The inner surface of the glass vessels and impellers were siliconized to prevent undue attachment of cells. Media: 10% FBS DMEM (Lonza, Walkersville, MD, USA) and PPRF-msc6. Cell seeding density: Static culture: Primary culture: 37, ,000 BM MNCs/cm 2 Passaged culture: 5,000 hmscs/cm 2 Stirred microcarrier culture: 24,000 cells/ml, based on a microcarrier loading density of 2 g Cytodex 3 microcarriers (GE Healthcare, Montreal, Quebec, Canada) per liter and a cell-to-bead ratio of 4:1. Table 3 Comparison of the number of T-flasks (225 cm 2 ) and stirred bioreactors, the amount of medium, and the time required for the generation of hmscs cultured in different culture methods 10% FBS DMEM + T-flasks, PPRF-msc6 + T-flasks, and PPRF-msc6 + microcarrier-based stirred bioreactor Passage No. P0 P1 P2 P3 P4 10% FBS DMEM hmscs obtained ( 10 6 ) Vessels needed 1 T-75 1 T T T T-225 Medium needed (ml) ,152 5,328 Time (days) PPRF-msc6 hmscs obtained ( 10 6 ) Vessels needed 1 T-75 2 T T-225 Medium needed (ml) ,304 Time (days) PPRF-msc6 + Cytodex 3 hmscs obtained ( 10 6 ) >200 a Vessels needed 1 T-75 2 T STR (>1.0 L) Medium needed (ml) >1,000 Time (days) a The number of hmscs to be generated by the use of instrumented stirred-tank reactors (STRs) was estimated, based on the data obtained from a microcarrier culture protocol not yet optimized. It is therefore expected that cell production yields per bioreactor unit, as well as the consistency of microcarrier culture performance, could further be increased by establishing a standard protocol with optimized culture variables. 116 Biotechnology and Applied Biochemistry

12 Media feeding: Static culture: Primary culture: Medium change (100%) was performed after 2 days to remove nonadherent cells, and thereafter 50% of medium was replenished every other day. Passaged culture: No medium change was performed. Stirred microcarrier culture: No medium change was performed when 2 g/l of Cytodex 3 microcarriers was used. The feeding strategy should be considered if the microcarriers are used at higher concentrations. Culture environment: T-flasks and spinner flasks were incubated at 37 Cina humidified atmosphere with 5% CO 2 (i.e., 20% O 2 ). Bench-top STRs were equipped with temperature, ph, and oxygen probes, supplied with mixed air via headspace aeration, and controlled by a computer to maintain the environmental parameters at predetermined set points (Fig. 4). The parameters and set points are as follows: Temperature (37 C): A heating jacket surrounding the vessel was used to control the temperature. ph (7.4): Carbon dioxide was supplied to the culture in order to maintain the medium ph through its interaction with sodium bicarbonate in the medium. Carbon dioxide supplied at gradually decreased concentrations (starting at 5.5% in air) was sufficient to maintain the predetermined set point throughout the entire culture period without the addition of extra acid/base. Fig. 4. Photographs of 500 ml DASGIP computer-controlled, stirred bioreactors. (A) Experimental setup for the bioreactor system showing: (1) instrumented bioreactor, (2) temperature and agitation module TC4SC4, (3) gas mixing station MX 4/4, (4) multipump modules MP8, (5) DASGIP control and data acquisition computer, and (6) DASGIP sensor module PH8PO8. (B) A closer view of the instrumented bioreactor, showing: (7) gas inlet to the bioreactor connected to O 2,N 2,CO 2, and air-gas cylinders via gas mixing station MX 4/4, (8) ph probe, (9) gas outlet from bioreactor, (10) heating jacket, (11) stirring plate, (12) dissolved O 2 probe, (13) temperature probe, and (14) bioreactor sampling port. Adapted with permission from InTech, ref. [103]. Dissolved oxygen concentration (70%): This value was readily maintained with a continuous supply of 17% oxygen in the mixed air. Subculture procedure: The primary cultures were harvested when well-developed colonies were formed, and the passaged cultures were harvested when cell growth reached 50% 80% confluence. To separate from the culture surface, the adherent cells were treated with trypsin EDTA. Typically, it is more difficult to culture primary cells compared with passaged cells. Indeed, several groups made unsuccessful attempts to develop defined serum-free media for the primary culture of MSCs [51 53],[104],[105]. It is therefore likely that the use of advanced bioreactor technologies such as the stirred microcarrier culture is not practical to support the attachment and growth of primary hmscs because the complex interactions of many mechanical, nutritional, and physiochemical variables would make it very complicated to find proper primary culture conditions. Moreover, as the frequency of hmscs in the initial primary cell populations is quite low, the mixed cells are typically inoculated at high concentrations (e.g., 150,000 cells/cm 2 ). Therefore, a T-75 flask should be enough to start the primary culture with the small starting cell population ( MNCs). When cultured in 10% FBS DMEM, this resulted in the generation of hmscs after an average of 13.5 days that is, PDs were ± 0.99 (n = 6) when the frequency of hmscs in the primary BM MNCs was assumed to be 0.001%. Subsequently, the derived hmscs were serially passaged using T-flasks. We demonstrated that the growth rate of cells from each donor BM was sustained >10 passages [55]. The cell-fold expansion of three donor cells was 4.69 ± 1.19 per passage, and cells were passaged every 3.73 days. As a result, hmscs were produced at P4 after 4 weeks (Table 3). After the primary culture, a total of cm 2 T-flasks were used with 6.8 L of medium (72 ml per T-225). Similarly, Bartmann et al. [71] generated over hmscs from small BM aspirates using 10% FBS-supplemented medium over a 4-week culture period. Unlike our protocol using a high-plating density for subculture ( 5,000 cells/cm 2 ), they used a two-step procedure to generate the clinical number of cells at P1, by plating the primary culture-derived cells at a low density (28 40 cells/cm 2 ) into 10 four-layered cell factories and culturing over 2 weeks. We assumed that over 16 L of medium (200 ml per layer, one-third medium change twice weekly) was used for this step. As described earlier, PPRF-msc6 supports a significantly greater cell expansion in primary BM culture. Namely, under the same primary culture condition described earlier, the use of PPRF-msc6 resulted in the generation of hmscs from MNCs in a T-75 flask within an average of 11.6 days, achieving ± 1.49 PDs (n = 8). Moreover, in the passaged cultures, the sustained rapid growth with negligible lag phase, together with the small, elongated morphology of cells in PPRF-msc6, led to a significantly high cell-fold increase at each passage. Specifically, we passaged hmscs from three donor BM cells in PPRF-msc6 at subconfluence every 3.5 days, obtaining ± 1.86 cell-fold expansion per passage. Using PPRF-msc6, therefore, we were able to generate over 500 Large-scale production of hmscs 117